Normal biological activity in a living organism combines endogenous expression of genes that constitute an individual's genome with responses to the outside world. In higher eukaryotes, gene expression begins in the nucleus with transcription of genomic DNA into a pre-mRNA or “primary” RNA transcript. While still in the nucleus, the pre-mRNA is modified to include a 5′ cap structure, forms heteronuclear ribonucleoprotein (hnRNP) complexes, acquires a 3′ polyadenylate tail and undergoes splicing to remove intervening RNA sequences (e.g. introns). The mature mRNA is then exported to the cytoplasm where protein complexes direct (1) association with ribosomes via the 5′ cap structure, termed Cap-dependent translation, or (2) interaction with cytosolic RNA binding proteins that facilitate mRNA storage, processing or degradation. Following ribosome-driven translation, sequential shortening of the 3′-polyadenylate tail results in transport of the mRNA body to a complex of ribonucleases (RNAses), termed the exosome, which degrades the aged mRNA and effectively terminates protein synthesis.
As expected, gene expression is a highly regulated process that must produce a desired gene product (typically a polypeptide) at a particular time, rate and quantity. In addition to transcriptional regulation, post-transcriptional processes such as mRNA decay and translation are key checkpoints in gene expression. It is not surprising that changes in a cellular expression profile, produced by genetic mutations or aberrant responses to external stimuli can cause severe abnormalities that often result in acute cell death or the manifestation of a chronic disease phenotype.
Extensive or prolonged cellular stimulation by environmental factors, such as altered nutrient levels, cytokines, hormones and temperature shifts, as well as environmental stresses like hypoxia, hypocalcemia, viral infection and tissue injury, results in the rapid attenuation of cap-dependent translation. This process is adaptive as it curtails global protein synthesis which is not needed for an immediate stress response and recovery. However, this translational abatement does not completely eliminate ribosome activity, since many products of stress response and recovery genes continue to be synthesized by an alternative process, termed cap-independent translation (reviewed in Guhaniyogi & Brewer, 2001, Gene 265 (1-2):11-23).
Cap-independent translation occurs by direct recruitment of ribosomes to specific RNA structures termed Internal Ribosome Entry Sites (IRESs). IRES elements have been identified in a number of eukaryotic mRNAs (Bonnal S et al., (2003) Nucleic Acids Res. 31:427-428) and ensure the efficient expression of proteins or fragments thereof during nuclear inactivity or acute cellular stress when “cap-dependent” translation initiation is inhibited (i.e., apoptosis, starvation, gamma-irradiation, hypoxia, mitosis, or terminal differentiation).
Bypassing the requirement for a 5′ mRNA cap structure was initially described as a mechanism for translating viral RNAs irrespective of a near complete inhibition of cellular cap-dependent translation in infected cells (Jang et al., 1988, J. Virol., 62:2636-43). Generally, IRES sequences cannot be identified by sequence homology and well characterized IRES elements have been verified using functional assays (Mountford and Smith, 1995, TIG, 11(5): 179-184; Baird et al., 2006, NAR, 12(10):1755-85). Current evidence shows that the conformation of the IRES RNA and the binding of accessory proteins to specific mRNA sequences enable ribosome binding. In eukaryotic cells, IRES-directed translation has often been associated with ˜5′ untranslated regions (5′UTRs) of mRNAs that contain unusually long and thermodynamically stable RNA secondary structures with multiple short open reading frames (ORFs) that dramatically inhibit the initiation of ribosome-dependent translation. However, functional verification of IRES activity for many of these 5′UTR IRES elements has been complicated by the presence of transcriptional effector sequences cloned from the overlapping 5′ gene promoter. Attempts to employ these 5′UTR elements in IRES reporter vectors have been complicated by this residual background transcriptional activity which masks any translational regulation produced by these sequences. Thus, the cap-independent translation and its regulation are still highly unexplored, as are the systems which utilize translation in general as a readout tool.